RESUMEN
Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate L-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer D-dopa than for L-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.
Asunto(s)
Dihidroxifenilalanina/química , Monofenol Monooxigenasa/química , Agaricales/enzimología , Catálisis , Dominio Catalítico , Catecol Oxidasa/química , Cinética , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Modelos Químicos , Oxígeno/química , Unión Proteica , Espectrofotometría/métodos , Factores de TiempoRESUMEN
There is controversy in the literature concerning the action of tetrahydropterines on the enzyme tyrosinase and on melanogenesis in general. In this study, we demonstrate that tetrahydropterines can inhibit melanogenesis in several ways: i) by non-enzymatic inhibition involving purely chemical reactions reducing o-dopaquinone to L-dopa, ii) by acting as substrates which compete with L-tyr and L-dopa, since they are substrates of tyrosinase; and iii) by irreversibly inhibiting the enzymatic forms met-tyrosinase and deoxy-tyrosinase in anaerobic conditions. Three tetrahydropterines have been kinetically characterised as tyrosinase substrates: 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin, 6-methyl-5,6,7,8-tetrahydropterine and 6,7-(R,S)-dimethyl-5,6,7,8-tetrahydropterine. A kinetic reaction mechanism is proposed to explain the oxidation of these compounds by tyrosinase.
Asunto(s)
Melaninas/biosíntesis , Monofenol Monooxigenasa/antagonistas & inhibidores , Pterinas/farmacología , Agaricales/enzimología , Unión Competitiva , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Levodopa/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidación-Reducción , Pterinas/química , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
BACKGROUND: Recently, the quantification of mitoses in cutaneous melanoma has been discharged from the main prognostic variables of the TNM classification. OBJECTIVE: To investigate the prognostic value of the presence of mitoses in primary cutaneous melanoma and to establish the number of mitoses per mm2 that may have prognostic significance. METHODS: A retrospective observational study was performed on 141 patients treated for cutaneous melanoma, who were assessed by the same pathologist, and who had a minimum follow-up of 2 years. Clinical, epidemiological, histopathological and follow-up variables were gathered and compared with the number of mitoses to distinguish the significance of differences by means of univariate, multivariate, and survival analyses. RESULTS: The cut-off level related to a better sensitivity and specificity was 1.50 mitoses per mm2. The presence of two or more mitoses/mm2 showed a better relationship with prognostic variables and both the overall and disease-free survival than the presence of 1 or more mitoses/mm2. This happens especially in melanomas thicker than 0.8 mm and it could affect the staging in cases with Breslow between 1 and 2 mm. CONCLUSIONS: A mitotic rate of two or more mitoses per mm2 in cutaneous melanoma should be considered as a more accurate prognostic factor than one or more mitoses per mm2, particularly in tumors equal or greater than 0.8 mm in thickness.
Asunto(s)
Melanoma/mortalidad , Melanoma/patología , Índice Mitótico , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Niño , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias/normas , Pronóstico , Curva ROC , Valores de Referencia , Estudios Retrospectivos , Sensibilidad y Especificidad , Biopsia del Ganglio Linfático Centinela , Análisis de Supervivencia , Adulto Joven , Melanoma Cutáneo MalignoRESUMEN
Tyrosinase is a copper enzyme with broad substrate specifity toward a lot of phenols with different biotechnological applications. The availability of quick and reliable measurement methods of the enzymatic activity of tyrosinase is of outstanding interest. A series of spectrophotometric methods for determining the monophenolase and diphenolase activities of tyrosinase are discussed. The product of both reactions is the o-quinone of the corresponding monophenol/diphenol. According to the stability and properties of the o-quinone, the substrate is classified as four substrate types. For each of these substrate types, we indicate the best method for measuring diphenolase activity (among eight methods) and, when applicable, for measuring monophenolase activity (among four methods). The analytical and numerical solutions to the system of differential equations corresponding to the reaction mechanism of each case confirm the underlying validity of the different spectrophotometric methods proposed for the kinetic characterization of tyrosinase in its action on different substrates.
Asunto(s)
Catecol Oxidasa/análisis , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/análisis , Espectrofotometría/métodos , Catecol Oxidasa/metabolismo , Cinética , Oxidorreductasas/metabolismo , Especificidad por SustratoRESUMEN
Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.
Asunto(s)
Catecol Oxidasa/metabolismo , Ácido Clorogénico/metabolismo , Peroxidasa/metabolismo , Quinonas/metabolismo , Agaricales/enzimología , Armoracia/enzimología , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Ácido Peryódico/metabolismoRESUMEN
Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells.
Asunto(s)
Reparación del ADN , Epigénesis Genética , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Cromatina/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Dipiridamol/metabolismo , Femenino , Antagonistas del Ácido Fólico/farmacología , Histonas/metabolismo , Humanos , Células MCF-7 , Metilación/efectos de los fármacos , Metilación/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
During the course of cancer progression, neoplastic cells undergo dynamic and reversible transitions between multiple phenotypic states, and this plasticity is enabled by underlying shifts in epigenetic regulation. Our results identified a negative feedback loop in which SET9 controls DNA methyltransferase-1 protein stability, which represses the transcriptional activity of the SET9 promoter in coordination with Snail. The modulation of SET9 expression in breast cancer cells revealed a connection with E2F1 and the silencing of SET9 was sufficient to complete an epigenetic program that favored epithelial-mesenchymal transition and the generation of cancer stem cells, indicating that SET9 plays a role in modulating breast cancer metastasis. SET9 expression levels were significantly higher in samples from patients with pathological complete remission than in samples from patients with disease recurrence, which indicates that SET9 acts as a tumor suppressor in breast cancer and that its expression may serve as a prognostic marker for malignancy.
Asunto(s)
Biomarcadores de Tumor , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , N-Metiltransferasa de Histona-Lisina/genética , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Análisis por Conglomerados , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Modelos Biológicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Pronóstico , Unión Proteica , Curva ROC , Factores de Transcripción de la Familia Snail/metabolismoRESUMEN
This paper deals with the quantitative description of the regulatory effect of pH on the oxidation pathway of L-dopa to yield melanins. Tyrosinase catalyzes the oxidation by molecular oxygen of L-dopa to o-dopaquinone, which evolves non-enzymatically through a branched pathway with cyclization or hydroxylation reactions. The production of several quinones and semiquinones in the pathway has also been reported. The intermediates of the hydroxylation branch have been identified and the corresponding rate constants have been determined. These compounds, such as have been detected in melanosomes and in tumoral cells, have great cytotoxic power and could have physiological significance in acidic media.
Asunto(s)
Levodopa/metabolismo , Melaninas/biosíntesis , Basidiomycota/enzimología , Concentración de Iones de Hidrógeno , Cinética , Matemática , Modelos Teóricos , Monofenol Monooxigenasa/metabolismo , EspectrofotometríaRESUMEN
The transient phase of tyrosinase activity acting on monophenols has been investigated. Although an analytical solution for the lag period (tau) cannot be obtained, its dependence on reagent concentration and pH is studied. It is established that decreases as the quantity of enzyme increases, although it increases when monophenol or pH are increased. The computer simulation shows those rate constants whose variations affect the transient phase most significantly. In addition, the steady state of the pathway is studied using tyrosinases from several sources such as mushroom, frog epidermis and grape. The kinetic analysis, which is based on not imposing restrictions on the values of the rate constants involved in the mechanism, allows us to obtain analytical expressions for both monophenolase and diphenolase activities and explains the experimental results obtained with the different enzymes. The values determined for the kinetic parameter, R, point to the monophenol hydroxylation step as being the limiting step of the turnover, while the values obtained for n suggest the absence of fast equilibrium in the oxidation of diphenol by Emet.
Asunto(s)
Monofenol Monooxigenasa/química , Secuencia de Aminoácidos , Animales , Basidiomycota , Concentración de Iones de Hidrógeno , Cinética , Matemática , Monofenol Monooxigenasa/metabolismo , Fenoles/metabolismo , Rana esculentaRESUMEN
The effect of ferrous ions on the monophenolase activity of tyrosinase has been studied. Although a shortening of the lag period which characterizes this hydroxylation reaction was observed, no direct effect on the enzyme was found. The reaction between ferrous ions and molecular oxygen in the presence of chelating agents, such as phosphate or EDTA, produces hydroxyl radicals. These radicals can hydroxylate tyrosine to generate L-3,4-dihydroxyphenylalanine (dopa). Catalase and scavengers of hydroxyl radicals inhibited both the shortening of the lag period and dopa formation. On the basis of these results, it is proposed that the influence of ferrous ions on tyrosinase is due to the formation of dopa in the chemical hydroxylation of tyrosine. Dopa transforms the Emet form of the enzyme (Cu2+Cu2+) into the Edeoxy form (Cu1+Cu1+) and, thus, shortens the lag period.
Asunto(s)
Dihidroxifenilalanina/biosíntesis , Compuestos Ferrosos/farmacología , Indolquinonas , Monofenol Monooxigenasa/efectos de los fármacos , Tirosina/metabolismo , Ácido Edético/farmacología , Radicales Libres , Hidroxilación , Indoles/metabolismo , Modelos Químicos , Oxígeno/metabolismo , Fosfatos/farmacología , Quinonas/metabolismo , Factores de TiempoRESUMEN
One basic peroxidase isoenzyme, with a pI of 8.8, is present in the intercellular washing fluid in the aerial part of 6-day-old Lupinus albus hypocotyl seedlings. This isoenzyme, called LuP-B2, is the principal soluble component secreted into the apoplastic space and it is a constitutive enzyme along the whole length of etiolated hypocotyl. The enzymatic inactivation process which this apoplastic peroxidase undergoes is described for the first time. The kinetic constants which describe its inactivation by H(2)O(2) in the absence of reductant substrates are determined. LuP-B2 is inactivated in situ and in vitro in a time- and concentration-dependent manner. H(2)O(2) acts as a suicide substrate according to a model previously proposed by us. The constant values calculated are similar to those calculated for the basic isoenzyme of horseradish roots, HRP-C. LuP-B2 presents a k(inact) value of 7.5 x 10(-3) s(-1) and a k(cat) of 6.7 s(-1). This isoenzyme makes 889 catalytic cycles for each inactivation event. The similarity in behavior and the constant values, together with other situations (both are excreted, soluble and constitutive isoenzymes) suggest that the inactivation process could play an important role in plant development and stress situations.
Asunto(s)
Hipocótilo/enzimología , Peroxidasas/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Peróxido de Hidrógeno/química , Focalización Isoeléctrica , Cinética , Peroxidasas/química , Peroxidasas/aislamiento & purificaciónRESUMEN
The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.
Asunto(s)
Dihidroxifenilalanina/análogos & derivados , Monofenol Monooxigenasa/metabolismo , Animales , Dihidroxifenilalanina/química , Dihidroxifenilalanina/metabolismo , Cinética , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period.
Asunto(s)
Agaricales/enzimología , Monofenol Monooxigenasa/química , Fenoles/química , Quinonas/química , Catecol Oxidasa/química , Relación Dosis-Respuesta a Droga , Cinética , Levodopa/química , Modelos Químicos , Modelos Teóricos , Monofenol Monooxigenasa/farmacología , Espectrofotometría , Tirosina/químicaRESUMEN
Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.
Asunto(s)
Catecoles/química , Monofenol Monooxigenasa/química , Fenoles/química , Agaricales , Benzoquinonas/química , Catecoles/farmacología , Cisteína/química , Cisteína/farmacología , Cromatografía de Gases y Espectrometría de Masas/métodos , Cinética , Melanosomas/metabolismo , Modelos Químicos , Modelos Teóricos , Monofenol Monooxigenasa/metabolismo , Oxidación-ReducciónRESUMEN
It has long been considered that ecto-5'-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4. 2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS-PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of beta-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.
Asunto(s)
5'-Nucleotidasa/química , Compuestos de Sulfhidrilo/química , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Cromatografía Líquida de Alta Presión , Dimerización , Ditiotreitol , Electroforesis en Gel de Poliacrilamida , Guanidina , Masculino , Estructura Cuaternaria de Proteína , UreaRESUMEN
Tyrosinase usually catalyzes the conversion of monophenols to o-diphenols and the oxidation of o-diphenols to the corresponding quinones. However, when 3,4-dihydroxymandelic acid was provided as the substrate, 3,4-dihydroxybenzaldehyde was produced. These results led to the proposal that tyrosinase catalyzes an unusual oxidative decarboxylation of this substrate (Sugumaran, M. (1986) Biochemistry 25, 4489-4492). However, 3,4-dihydroxybenzaldehyde is also obtained through the oxidation of 3,4-dihydroxymandelic acid by sodium periodate and on a mercury electrode. These results led to the proposal that tyrosinase catalyzes the oxidation of the substrate into o-quinone, which reacts immediately with a molecule of substrate, oxidizing it and through decarboxylation generates an intermediate (quinone methide) which transforms into 3,4-dihydroxybenzaldehyde; simultaneously, the original o-quinone is reduced to 3,4-dihydroxymandelic acid.
Asunto(s)
Catecol Oxidasa/metabolismo , Ácidos Mandélicos/metabolismo , Monofenol Monooxigenasa/metabolismo , Basidiomycota/enzimología , Oxidación-Reducción , Ácido Peryódico , Polarografía , Análisis EspectralRESUMEN
Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.
Asunto(s)
Acetilcisteína/análisis , Catecol Oxidasa , Cisteína/análisis , Espectrofotometría , Agaricales/enzimología , Catecol Oxidasa/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Cancer is characterized by uncontrolled cell growth and the acquisition of metastatic properties. In most cases, the activation of oncogenes and/or deactivation of tumour suppressor genes lead to uncontrolled cell cycle progression and inactivation of apoptotic mechanisms. Although the underlying mechanisms of carcinogenesis remain unknown, increasing evidence links aberrant regulation of methylation to tumourigenesis. In addition to the methylation of DNA and histones, methylation of nonhistone proteins, such as transcription factors, is also implicated in the biology and development of cancer. Because the metabolic cycling of methionine is a key pathway for many of these methylating reactions, strategies to target the epigenetic machinery of cancer cells could result in novel and efficient anticancer therapies. The application of these new epigenetic therapies could be of utility in the promotion of E2F1-dependent apoptosis in cancer cells, in avoiding metastatic pathways and/or in sensitizing tumour cells to radiotherapy.
Asunto(s)
Terapia Genética/métodos , Neoplasias/genética , Neoplasias/terapia , Animales , Metilación de ADN , Epigenómica , HumanosRESUMEN
Many studies concerning the effect of ascorbic acid on the action of tyrosinase on several substrates have been carried out with contradictory results. The results shown in this work comprise a hypothetical reaction mechanism, which explains the ascorbate oxidase activity of frog epidermis tyrosinase. The reaction between frog epidermis tyrosinase and L-ascorbic acid was studied by oxymetric and spectrophotometric assays. The activity was linearly related to enzyme concentration, with a Michaelis constant for L-ascorbic acid of 0.160 +/- 0.009 mM and Vmax of 90 +/- 4 nM/s. Maximum activity was obtained at pH 7.5. The stoichiometry of the reaction was calculated by measuring the substrate (O2 and L-ascorbic acid) consumption as well as the initial rates of the consumption of oxygen and the disappearance of L-ascorbic acid. The stoichiometry was found to be 1:2 (O2:L-ascorbic acid). The action of the tyrosinase inhibitor tropolone was also studied. All the results present evidence concerning the ascorbate oxidase activity of frog epidermis tyrosinase and a possible reaction mechanism based on the different enzymatic forms of tyrosinase to explain such activity.
Asunto(s)
Ascorbato Oxidasa/metabolismo , Ácido Ascórbico/metabolismo , Epidermis/enzimología , Monofenol Monooxigenasa/metabolismo , Animales , Anuros , Activación Enzimática , Especificidad por SustratoRESUMEN
The kinetic behaviour of tyrosinase is very complex because the enzymatic oxidation of monophenol and o-diphenol to o-quinones occurs simultaneously with the coupled non-enzymatic reactions of the latter. Both reaction types are included in the kinetic mechanism proposed for tyrosinase (Mechanism I [J. Biol. Chem. 267 (1992) 3801-3810]). We previously confirmed the validity of the rate equations by the oxidation of numerous monophenols and o-diphenols catalysed by tyrosinase from different fruits and vegetables. Other authors have proposed a simplified reaction mechanism for tyrosinase (Mechanism II [Theor. Biol. 203 (2000) 1-12]), although without deducing the rate equations. In this paper, we report new experimental work that provides the lag period value, the steady-state rate, o-diphenol concentration released to the reaction medium. The contrast between these experimental data and the respective numerical simulations of both mechanisms demonstrates the feasibility of Mechanism I. The need for the steps omitted from Mechanism II to interpret the experimental data for tyrosinase, based on the rate equations previously deduced for Mechanism I is explained.